Human mast cell-expressed membrane proteins

ABSTRACT

Mast cell-expressed membrane proteins that are highly expressed in human mast cells and the lungs as compared to brain, heart, kidney, liver, tracheal, and similar tissues. The proteins are transmembrane proteins involved in the regulation of mast cell and lung tissue function, including degranulation of mast cells. The preferred protein is a 187 amino acid transmembrane protein having an intercellular domain comprising amino acids  1  through  82,  a transmembrane domain comprising amino acids  83  through  105,  and an extracellular domain comprising amino acids  106  through  187  (SEQ ID NO:2).

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/345,909, filed Jan. 3, 2002, the disclosure of which isincorporated herein by this reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to cellular membrane proteins andparticularly to mast cell-expressed membrane proteins (“MCEMP(s)”).

2. Description of the Prior Art

Mast cells originate from hematopoietic stem cells in the bone marrowbut complete their development only after they migrate into diverseperipheral tissues. Mature mast cells express a high-affinity IgEreceptor known as FcεRI on their surface. FcεRI can be activated byreceptor bound IgE that has been cross-linked with specific allergens.Mast cells can also be activated by IgE independent mechanisms. Forexample, complement proteins C3a and C5a have been shown to activatemast cells in vivo and calcium ionophores, such as A23187, have beenshown to activate mast cells in vitro.

Mast cells contain a wide variety of preformed secretory inflammatorymediators such as histamine, tryptase, proteases, peroxidase, andneutrophil chemotactic factor. Upon activation, mast cells release thesepreformed mediators and certain newly synthesized lipid mediators suchas arachidonic acid metabolites (leukotrienes), prostaglandins, andcytokines into the surrounding tissues. Typically, the cells releaseboth induced immunomodulatory and proinflammatory cytokines, e.g., TNFα,IL4, IL-13, IL-5, IL-10, and chemokines.

It is well known that human mast cells play a critical role in thepathogenesis of many inflammatory and allergic diseases such as asthmaand atopic dermatitis. The preformed and newly synthesized mediatorsreleased by mast cells are responsible for most of the early events inallergic reactions and, through cytokine production and othermechanisms, contribute to the expression of late-phase reactions andchronic allergic inflammation. Mast cells have also been observed in amultitude of neoplastic, fibrotic, and inflammatory processes such aslymphoproliferative disorders, interstitial lung disease, and thesynovium in rheumatoid arthritis. Furthermore, the number of mast cellsis highly elevated in other inflammatory diseases such as inflammatorybowel disease. Mast cells also play a role in the progression of heartfailure. During heart failure, mast cells are found in the human heartin increased numbers and their density is higher in ischemiccardiomyopathy. U.S. Pat. No. 6,140,348 discloses a method forpreventing and treating heart failure by inhibiting mast celldegranulation. Mast cells also play an important role in multiplesclerosis. Mast cell specific genes were found in the brain lesions ofmultiple sclerosis and a mast cell stabilizer was found to amelioratethe severity of experimental allergic encephalomyelitis (EAE), theanimal model of multiple sclerosis.

Since mast cells play such a critical role in allergic and otherinflammatory diseases, drugs or other agents that regulate mast celldifferentiation, proliferation, adhesion, maturation, activation, anddegranulation may be of use to prevent or treat such diseases.Therefore, there is a need to identify novel mast cell proteins thatplay a role in mast cell mediated diseases and to develop drugs andmethods for regulating mast cell activity.

SUMMARY OF THE INVENTION

It is, therefore, an object of the invention to provide novel mastcell-expressed membrane proteins (“MCEMP(s)”) involved in regulatingmast cell activity.

It is another object of the invention to provide agonists or antagoniststhat bind to MCEMPs and their ligands and regulate their function andactivity.

It is another object of the invention to provide antibodies that bind toMCEMPs and methods for producing such antibodies.

It is further object of the invention to provide nucleotide sequencesthat encode novel MCEMPs.

It is another object of the invention to provide vectors comprisingnucleotide sequences that encode novel MCEMPs and host cells containingsuch vectors.

It is a further object of the invention to provide a screening methodfor identifying MCEMP agonists and antagonists and for determiningwhether pharmaceuticals are likely to cause undesirable side effectswhen administered to an animal.

It is another object of the present invention to provide a method forblocking or modulating the expression of MCEMPs.

It is another object of the present invention to provide a method fordiagnosing the predisposition of a mammal to develop diseases caused bythe unwanted MCEMP activity.

It is a further object of the invention to provide a method forpreventing or treating MCEMP mediated diseases in a mammal.

It is another object of the present invention to provide a diagnosticmethod for detecting MCEMPs expressed by specific cells, tissues, orbody fluids.

It is another object of the present invention to provide a method forisolating and purifying MCEMPs from recombinant cell culture,contaminants, and native environments.

It is a further object of the present invention to provide vaccines andmethods for vaccinating a mammal against MCEMP meditated diseases.

These and other objects are achieved by providing a novel MCEMP havingthe amino sequence shown in SEQ ID NO:2, the nucleotide sequence thatcodes for the protein, and the vectors and host cells that express thenucleotide sequence and produce the protein. The MCEMP is used toproduce agonist and antagonist antibodies useful for affecting mast cellfunction such as degranulation, adhesion, migration, apoptosis, and therelease of mast cell mediators. The antibodies are useful for screeningfor MCEMP agonists and antagonists and for screening pharmaceuticals todetermine if they are likely to cause undesirable side effects whenadministered to an animal for medicinal purposes.

Other and further objects, features, and advantages of the presentinvention will be readily apparent to those skilled in the art.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “purified polypeptide” means a polypeptide identified andseparated from at least one contaminant polypeptide ordinarilyassociated with the purified polypeptide in its native environment,particularly a polypeptide separated from its cellular environment.

The term “isolated polynucleotide” means a polynucleotide identified andseparated from at least one contaminant polynucleotide ordinarilyassociated with the isolated polynucleotide in its native environment,particularly a polynucleotide separated from its cellular environment.

The term “native” when used to describe a polynucleotide, polypeptidesequence, or other molecule means a polypeptide, polynucleotide, orother molecule as found in nature, e.g., a polypeptide or polynucleotidcsequence that is present in an organism such as a virus or prokaryoticor eukaryotic cell that can be isolated from a source in nature and thathas not been intentionally modified to change is structure, properties,or function. An unisolated cellular polynucleotide having the nucleotidesequence shown in SEQ ID NO:1 is a native polynucleotide and unpurifiedcellular polypeptide having the amino acid sequence shown in SEQ ID NO:2is a native polypeptide.

The term “percent sequence identity” means the percentage of sequencesimilarity found in a comparison of two or more nucleotide or amino acidsequences. Percent identity can be determined electronically, e.g., byusing the MEGALIGN program (DNASTAR, Inc., Madison Wis.). The MEGALIGNprogram creates alignments between two or more sequences according todifferent methods, e.g., the clustal method. (See, e.g., Higgins, D. G.and P. M. Sharp (1988) Gene 73:237-244.) The clustal algorithm groupssequences into clusters by examining the distances between all pairs.The clusters are aligned pairwise and then in groups. The percentagesimilarity between two amino acid sequences, e.g., sequence A andsequence B, is calculated by dividing the length of sequence A, minusthe number of gap residues in sequence A, minus the number of gapresidues in sequence B, into the sum of the residue matches betweensequence A and sequence B, times one hundred. Gaps of low or of nosimilarity between the two amino acid sequences are not included indetermining percentage similarity. Percent identity between nucleotidesequences is counted or calculated by methods known in the art, e.g.,the Jotun Hein method given in Hein, J. (1990) Methods Enzymol.183:626-645. Identity between sequences can also be determined by othermethods known in the art, e.g., by varying hybridization conditions.

The term “variant” when used to describe a polynucleotide sequence meansa nucleotide sequence that differs from its native counterpart by one ormore nucleotides and either has the same or similar biological functionas its native counterpart or does not have the same or similarbiological function as its native counterpart but is useful as a probeto identify or isolate its native counterpart. Preferred variants arenucleotide sequences having at least 85 percent sequence identity whencompared to its native counterpart, preferably at least 90 to 95 percentsequence identity, and most preferably at least 99 percent sequenceidentity, and nucleotide sequences that bind to native sequences ortheir complement under stringent conditions. Most Preferred variants arenucleotide sequences that code for the same amino acid sequence as itsnative counterpart but differ from the native nucleotide sequence basedonly on the degeneracy of the genetic code.

The term “variant” when used to describe a polypeptide sequence means anamino acid sequence that differs from its native counterpart by one ormore amino acids, including modifications, substitutions, insertions,and deletions, and either has the same or similar biological function asits native counterpart or does not have the same or similar biologicalfunction as its native counterpart but is useful as an immunogen toproduce antibodies that bind to its native counterpart or as an agonistor antagonist for its native counterpart. Preferred variants arepolypeptides having at least 70 percent sequence identity when comparedto its native counterpart, preferably at least 85 percent sequenceidentity, and most preferably at least 95 percent sequence identity.Most Preferred variants are polypeptides with conservative amino acidsubstitutions.

The term “fragment” when used to describe a polynucleotide means anucleotide sequence subset of its native counterpart that binds to itsnative counterpart or its complement under stringent conditions.Preferred fragments have a nucleotide sequence of at least 25 to 50consecutive nucleotides of the native sequence. Most preferred fragmentshave an amino acid sequence of at least 50 to 100 consecutivenucleotides of the native sequence.

The term “fragment” when used to describe a polypeptide means an aminoacid sequence subset of its native counterpart that either retains anybiological activity of its native counterpart or acts as an immunogencapable of producing an antibody that binds to its native counterpart.Preferred fragments have an amino acid sequence of at least 10 to 20consecutive amino acids of the native sequence. Most preferred fragmentshave an amino acid sequence of at least 20 to 30 consecutive amino acidsof the native sequence.

The term “agonist” means any molecule that promotes, enhances, orstimulates the normal function of the MCEMPs. One type of agonist is amolecule that interacts with a MCEMP in a way that mimics its ligand,including an antibody or antibody fragment.

The term “antagonist” means any molecule that blocks, prevents,inhibits, or neutralizes the normal function of the MCEMPs. One type ofantagonist is a molecule that interferes with the interaction betweenMCEMPs and its ligand, including an antibody or antibody fragment.Another type of antagonist is an antisense nucleotide that inhibitsproper transcription of native MCEMPs.

The term “conservative amino acid substitution” means that an amino acidin a polypeptide has been substituted for with an amino acid having asimilar side chain. For example, glycine, alanine, valine, leucine, andisoleucine have aliphatic side chains; serine and threonine havealiphatic-hydroxyl side chains; asparagine and glutamine haveamide-containing side chains; phenylalanine, tyrosine, and tryptophanhave aromatic side chains; lysine, arginine, and histidine have basicside chains; and cysteine and methionine have sulfur-containing sidechains. Preferred conservative amino acids substitutions arevaline-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

The term “stringent conditions” means (1) hybridization in 50% (vol/vol)formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1%polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750mM NaCl, 75 mM sodium citrate at 42° C., (2) hybridization in 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C.; with washes at 42° C. in 0.2×SSC and 0.1% SDS or washes with0.015 M NaCl, 0.0015 M sodium citrate, 0.1% Na₂SO₄ at 50° C. or similarprocedures employing similar low ionic strength and high temperaturewashing agents and similar denaturing agents.

The term “antisense” as used herein, refers to any compositioncontaining nucleotide sequences which are complementary to a specificDNA or RNA sequence. The term “antisense strand” is used in reference toa nucleic acid strand that is complementary to the “sense” strand.Antisense molecules include peptide nucleic acids and may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and block either transcription ortranslation. The designation “negative” is sometimes used in referenceto the antisense strand, and “positive” is sometimes used in referenceto the sense strand.

The term “knockout” refers to partial or complete reduction of theexpression of at least a portion of a polypeptide encoded by anendogenous gene (such as the gene for MCEMPs) of a single cell, selectedcells, or all of the cells of a mammal. The mammal may be a“heterozygous knockout” having one allele of the endogenous genedisrupted or “homozygous knockout” having both alleles of the endogenousgene disrupted.

The term “MCEMP(s)” means amino acid sequences of substantially purifiedMCEMPs obtained from any species, particularly mammalian, includingbovine, ovine, porcine, murine, equine, and preferably human, from anysource whether natural, synthetic, semi-synthetic, or recombinant.

This invention is not limited to the particular methodology, protocols,cell lines, vectors, and reagents described herein because they mayvary. Further, the terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to limit thescope of the present invention. As used herein and in the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise, e.g., reference to “ahost cell” includes a plurality of such host cells.

Because of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding the MCEMPs of the present invention may be produced.Some of these sequences will be highly homologous and some will beminimally homologous to the nucleotide sequences of any known andnaturally occurring nucleotide sequence. The present inventioncontemplates each and every possible variation of nucleotide sequencethat could be made by selecting combinations based on possible codonchoices. These combinations are made in accordance with the standardtriplet genetic code as applied to the nucleotide sequence that codesfor naturally occurring MCEMPs and all such variations are to beconsidered as being specifically disclosed.

Unless defined otherwise, all technical and scientific terms and anyacronyms used herein have the same meanings as commonly understood byone of ordinary skill in the art in the field of the invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice of the present invention, thepreferred methods, devices, and materials are described herein.

All patents and publications mentioned herein are incorporated herein byreference to the extent allowed by law for the purpose of describing anddisclosing the proteins, enzymes, vectors, host cells, and methodologiesreported therein that might be used with the present invention. However,nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

THE INVENTION Polypeptides

In one aspect, the present invention provides a purified polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:2; a variant of SEQ ID NO:2; a fragment of SEQ ID NO:2; anamino acid sequence encoded by an isolated polynucleotide comprising anucleotide sequence selected from the group consisting of SEQ ID NO:1; avariant of SEQ ID NO:1; and a fragment of SEQ ID NO:1.

The purified polypeptides of the present invention are mastcell-expressed membrane proteins (“MCEMP(s)”) that are highly expressedin human mast cells and the lungs. The proteins are transmembraneproteins involved in the regulation of mast cell and lung tissuefunction. In the preferred embodiment, the protein is a 187 amino acidprotein having the sequence shown in SEQ ID NO:2 (“MCEMP1”). Thepreferred protein has an intercellular domain comprising amino acids 1through 82, a transmembrane domain comprising amino acids 83 through105, and an extracellular domain comprising amino acids 106 through 187.

The polypeptides of the present invention are used to create antibodiesthat bind to the proteins and influence mast cell and lung cellstructure, properties, or function, including biological functions suchas degranulation, adhesion, migration, apoptosis, and the release ofmast cell contents. Preferably, the antibodies function as MCEMPagonists to activate the production of mast cell mediators or as MCEMPantagonists to inhibit the production of mast cell proinflammnatorymediators such as histamines, TNFα, and leukotrienes.

Agonists and Antagonists

In another aspect, the present invention provides agonists andantagonists that specifically bind to a MCEMP or its ligand and inhibitor activate its cellular function. Types of agonist and antagonistsinclude, but are not limited to, polypeptides, proteins, peptides,glycoproteins, glycopeptides, glycolipids, polysaccharides,oligosaccharides, nucleotides, organic molecules, bioorganic molecules,peptidomimetics, pharmacological agents and their metabolites, andtranscriptional and translation control sequences.

In one embodiment, the antagonists are a soluble form of MCEMP andsoluble polypeptides derived from the extracellular domains of MCEMPsthat are capable of interfering with the ability of a MCEMP to interactwith its natural ligand. Preferably, the antagonists are peptidesselected from the group consisting of amino acids 106 through 187 of SEQID NO:2 or antagonist fragments thereof. These antagonistic block thebinding of the natural ligand for MCEMPs by binding to the ligand andpreventing the ligand from binding to its native receptor.

Preferably, the agonists and antagonists are antibodies that bindspecifically to MCEMP and influence their biological actions andfunctions, e.g., to activate or inhibit degranulation and control therelease of mast cell mediators. The antibodies can be polyclonal ormonoclonal antibodies but are preferably monoclonal antibodies.

Antagonist antibodies are used to prevent or treat diseasescharacterized by the activation of mast cells, e.g., diseases caused bydegranulation and the release of mast cell contents. Agonist antibodiesare used to prevent or treat diseases characterized by relatively lowmast cell mediator concentration.

The agonists and antagonists are used for the treatment of variousimmune diseases, including, but not limited to allergic diseases such asasthma, allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria; transplantation associated diseases including graft rejectionand graft-versus-host-disease; autoimmune or immune-mediated skindiseases including bullous skin diseases, erythema multiforme andcontact dermatitis, psoriasis; rheumatoid arthritis, juvenile chronicarthritis; inflammatory bowel disease (i.e., ulcerative colitis, Crohn'sdisease); systemic lupus erythematosis; spondyloarthropathies; systemicsclerosis (seleroderma); idiopathic inflammatory myopathies(dermatomyositis, polymyositis); Sjogren's syndrome; systemicvasculitis; sarcoidosis; autoimmune hemolytic anemia (immunepancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmunethrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediatedthrombocytopenia); thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis);diabetes mellitus; immune-mediated renal disease (glomerulonephritis,tubulointerstitial nephritis); demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinatingpolyneuropathy or Guillain-Barre syndrome, and chronicinflammatory demyelinating polyneuropathy; hepatobiliary diseases suchas infectious hepatitis (hepatitis A, B, C, D, E and othernon-hepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis;inflammatory and fibrotic lung diseases such as cystic fibrosis,gluten-sensitive enteropathy, and Whipple's disease; immunologicdiseases of the lung such as eosinophilic pneumonia, idiopathicpulmonary fibrosis and hypersensitivity pneumonitis.

Antibody and Antibody Production

In another aspect, the present invention provides an antibody that bindsto the MCEMPs of the present invention and methods for producing suchantibody, including antibodies that function as MCEMP agonists orantagonists. In one embodiment, the method comprises using isolatedMCEMPs or antigenic fragments thereof as an antigen for producingantibodies that bind to the MCEMPs of the present invention in a knownprotocol for producing antibodies to antigens, including polyclonal andmonoclonal antibodies. In another embodiment, the method comprises usinghost cells that express recombinant MCEMPs as an antigen. In a furtherembodiment, the method comprises using DNA expression vectors containingthe MCEMP gene to express the MCEMP as an antigen for producing theantibodies.

Methods for producing antibodies, including polyclonal, monoclonal,monovalent, humanized, human, bispecific, and heteroconjugateantibodies, are well known to skilled artisans.

Polyclonal Antibodies

Polyclonal antibodies can be produced in a mammal by injecting animmunogen alone or in combination with an adjuvant. Typically, theimmunogen is injected in the mammal using one or more subcutaneous orintraperitoneal injections. The immunogen may include the polypeptide ofinterest or a fusion protein comprising the polypeptide and anotherpolypeptide known to be immunogenic in the mammal being immunized. Theimmunogen may also include cells expressing a recombinant MCEMP or a DNAexpression vector containing the MCEMP gene. Examples of suchimmunogenic proteins include, but are not limited to, keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants include, but are not limited to,Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

Monoclonal Antibodies

Monoclonal antibodies can be produced using hybridoma methods such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host mammal, isimmunized with an immunogen to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theimmunogen. Alternatively, the lymphocytes may be immunized in vitro. Theimmunogen will typically include the polypeptide of interest or a fusionprotein containing such polypeptide. Generally, peripheral bloodlymphocytes (“PBLs”) cells are used if cells of human origin aredesired. Spleen cells or lymph node cells are used if cells of non-humanmammalian origin are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, e.g., polyethyleneglycol, to form a hybridoma cell (Goding, Monoclonal Antibodies:Principles and Practice, pp 59-103 (Academic Press, 1986)). Immortalizedcell lines are usually transformed mammalian cells, particularly rodent,bovine, or human myeloma cells. Usually, rat or mouse myeloma cell linesare employed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT), the culture medium for the hybridomas typicallywill include hypoxanthine, aminopterin, and thymidine (HAT medium). TheHAT medium prevents the growth of HGPRT deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma linessuch as those derived from MOPC-21 and MPC-11 mouse tumors availablefrom the Salk Institute Cell Distribution Center, San Diego, Calif. USA,and SP2/0 or X63-Ag8-653 cells available from the American Type CultureCollection, Rockville, Md. USA. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for use in theproduction of human monoclonal antibodies (Kozbor, J. lmmunol. 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Themouse myeloma cell line NS0 may also be used (European Collection ofCell Cultures, Salisbury, Wiltshire UK). Human myeloma and mouse-humanheteromycloma cell lines, well known in the art, can also be used toproduce human monoclonal antibodies.

The culture medium used for culturing hybridoma cells can then beassayed for the presence of monoclonal antibodies directed against thepolypeptide of interest. Preferably, the binding specificity ofmonoclonal antibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, e.g.,radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem, 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods.Suitable culture media for this purpose include Dulbecco's ModifiedEagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cellsmay be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones are isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as protein A-Sepharose,hydroxylapatite chromatography, gel electrophoresis, dialysis, oraffinity chromatography.

The monoclonal antibodies may also be produced by recombinant DNAmethods, e.g., those described in U.S. Pat. No. 4,816,567. DNA encodingthe monoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures, e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies (Innis M. et al. In “PCRProtocols. A Guide to Methods and Applications”, Academic, San Diego,Calif. (1990), Sanger, F. S, et al. Proc. Nat. Acad. Sci. 74:5463-5467(1977)). The hybridoma cells described herein serve as a preferredsource of such DNA. Once isolated, the DNA may be placed into expressionvectors. The vectors are then transfected into host cells such as simianCOS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that donot otherwise produce immunoglobulin protein. The recombinant host cellsarc used to produce the desired monoclonal antibodies. The DNA also maybe modified, for example, by substituting the coding sequence for humanheavy and light chain constant domains in place of the homologous murinesequences or by covalently joining the immunoglobulin coding sequence toall or part of the coding sequence for a non-immunoglobulin polypeptide.Such a non-immunoglobulin polypeptide can be substituted for theconstant domains of an antibody or can be substituted for the variabledomains of one antigen combining site of an antibody to create achimeric bivalent antibody.

Monovalent antibodies can be produced using the recombinant expressionof an immunoglobulin light chain and modified heavy chain. The heavychain is truncated generally at any point in the Fc region so as toprevent heavy chain crosslinking. Alternatively, the relevant cysteineresidues are substituted with another amino acid residue or are deletedso as to prevent crosslinking. Similarly, in vitro methods can be usedfor producing monovalent antibodies. Antibody digestion can be used toproduce antibody fragments, preferably Fab fragments, using knownmethods.

Antibodies and antibody fragments can be produced using antibody phagelibraries generated using the techniques described in McCafferty, etal., Nature 348:552-554 (1990). Clackson, et al., Nature 352:624-628(1991) and Marks, et al., J. Mol. Biol. 222:581-597 (1991) describe theisolation of murine and human antibodies, respectively, using phagelibraries. Subsequent publications describe the production of highaffinity (nM range) human antibodies by chain shuffling (Marks, et al.,Bio/Technology 10:779-783 (1992)), as well as combinatorial infectionand in vivo recombination as a strategy for constructing very largephage libraries (Waterhouse, et al., Nuc. Acids. Res. 21:2265-2266(1993)). Thus, these techniques are viable alternatives to traditionalmonoclonal antibody hybridoma techniques for isolation of monoclonalantibodies. Also, the DNA may be modified, for example, by substitutingthe coding sequence for human heavy-chain and light-chain constantdomains in place of the homologous murine sequences (U.S. Pat. No.4,816,567; Morrison, et al., Proc. Nat. Acad. Sci. USA 81:6851 (1984)),or by covalently joining to the immunoglobulin coding sequence all orpart of the coding sequence for a non-immunoglobulin polypeptide.Typically, such ion-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

Antibodies can also be produced using use electrical fusion rather thanchemical fusion to form hybridomas. This technique is well established.Instead of fusion, one can also transform a B-cell to make it immortalusing, for example, an Epstein Barr Virus, or a transforming gene“Continuously Proliferating Human Cell Lines Synthesizing Antibody ofPredetermined Specificity,” Zurawaki, V. R. et al, in “MonoclonalAntibodies,” ed. by Kennett R. H. et al, Plenum Press, N.Y. 1980, pp19-33.

Humanized Antibodies

Humanized antibodies can be produced using the method described byWinter in Jones et al., Nature, 321:522-525 (1986); Riechmann et al.,Nature, 332:323-327 (1988); and Verhoeyen et al., Science, 239:1534-1536 (1988). Humanization is accomplished by substituting rodentCDRs or CDR sequences for the corresponding sequences of a humanantibody. Generally, a humanized antibody has one or more amino acidsintroduced into it from a source that is non-human. Such “humanized”antibodies are chimeric antibodies wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some CDR residues and possibly someFR residues are substituted by residues from analogous sites in rodentantibodies. Humanized forms of non-human (e.g., murine or bovine)antibodies are chimeric immunoglobulins, immunoglobulin chains, orimmunoglobulin fragments such as Fv, Fab, Fab′, F(ab′)₂, or otherantigen-binding subsequences of antibodies that contain minimal sequencederived from non-human immunoglobulin. Humanized antibodies includehuman immunoglobulins (recipient antibody) wherein residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat, or rabbit having the desired specificity, affinity, andcapacity. Sometimes, Fv framework residues of the human immunoglobulinare replaced by corresponding non-human residues. Humanized antibodiesalso comprise residues that are found neither in the recipient antibodynor in the imported CDR or framework sequences. In general, humanizedantibodies comprise substantially all of at least one and typically twovariable domains wherein all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. Humanized antibodies optimally comprise at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin.

Human Antibodies

Human antibodies can be produced using various techniques known in theart, e.g., phage display libraries as described in Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991) and Marks et al., J. Mol. Biol.,222:581 (1991). Human monoclonal antibodies can be produced using thetechniques described in Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Liss, p. 77 (1985) and Boemer et al., J. Immunol.,147(1):86-95 (1991). Alternatively, transgenic animals, e.g., mice, areavailable which, upon immunization, can produce a full repertoire ofhuman antibodies in the absence of endogenous immunoglobulin production.Such transgenic mice are available from Abgenix, Inc., Fremont, Calif.,and Medarex, Inc., Annandale, N.J. It has been described that thehomozygous deletion of the antibody heavy-chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. Transfer of the human germ-lineimmunoglobulin gene array in such germ-line mutant mice will result inthe production of human antibodies upon antigen challenge. See, e.g.,Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovitset al., Nature 362:255-258 (1993); Bruggermann et al., Year in Immunol.7:33 (1993); and Duchosal et al. Nature 355:258 (1992). Human antibodiescan also be derived from phage-display libraries (Hoogenboom et al., J.Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581-597(1991); Vaughan et al., Nature Biotech 14:309 (1996)).

Bispecific Antibodies

Bispecific antibodies can be produced by the recombinant co-expressionof two immunoglobulin heavy-chain/light-chain pairs wherein the twoheavy chains have different specificities. Bispecific antibodies aremonoclonal, preferably human or humanized, antibodies that have bindingspecificities for at least two different antigens. In the presentinvention, one of the binding specificities is for the MCEMP and theother is for any other antigen, preferably a cell surface receptor orreceptor subunit. Because of the random assortment of immunoglobulinheavy and light chains, these hybridomas produce a potential mixture often different antibodies. However, only one of these antibodies has thecorrect bispecific structure. The recovery and purification of thecorrect molecule is usually accomplished by affinity chromatography.

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy chain constant domain comprising at least part ofthe hinge, CH2, and CH3 regions. Preferably, the first heavy-chainconstant region (CH1) containing the site necessary for light-chainbinding is present in at least one of the fusions. DNAs encoding theimmunoglobulin heavy-chain and, if desired, the immunoglobulin lightchain is inserted into separate expression vectors and co-transfectedinto a suitable host organism. Suitable techniques are shown in forproducing bispecific antibodies are described in Suresh et al., Methodsin Enzymology, 121:210 (1986).

Heteroconjugate Antibodies

Heteroconjugate antibodies can be produced known protein fusion methods,e.g., by coupling the amine group of one an antibody to a thiol group onanother antibody or other polypeptide. If required, a thiol group can beintroduced using known methods. For example, immunotoxins comprising anantibody or antibody fragment and a polypeptide toxin can be producedusing a disulfide exchange reaction or by forming a thioether bond.Examples of suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate. Such antibodies can be used to targetimmune system cells to unwanted cells or to treat HIV infections.

Polynucleotides

In another aspect, the present invention provides an isolatedpolynucleotide comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NO:1; a variant of SEQ ID NO:1; a fragment of SEQID NO:1; a nucleotide sequence that encodes a polypeptide having theamino acid sequence selected from the group consisting of SEQ ID NO:2; avariant of SEQ ID NO:2; and a fragment of SEQ ID NO:2. In oneembodiment, the isolated polynucleotide comprises a nucleotide sequencethat encodes a polypeptide having an amino acid sequence selected fromthe group consisting of amino acids 106 to 187 of SEQ ID NO:2 orantagonist fragments thereof.

The isolated polynucleotides of the present invention are preferablycoding sequences for MCEMPs involved in the regulation of mast cell andlung function. The polynucleotides are used to produce MCEMPs thatfunction as antigens in the process used to produce the agonist andantagonist antibodies that specifically bind to MCEMPs and inhibit oractivate the degranulation of mast cells.

Vectors and Host Cells

In another aspect, the present invention provides a vector comprising anucleotide sequence encoding the MCEMPs of the present invention and ahost cell comprising such a vector. The vector may contain SEQ ID NO: 2or, in one embodiment, nucleotides 455 through 1018 of SEQ ID NO:1 incombination with any regulatory, expression, or other vector sequencesrequired to express MCEMPs.

By way of example, the host cells may be mammalian cells, (e.g. CHOcells), prokaryotic cells (e.g., E. coli) or yeast cells (e.g.,Saccharomyces cerevisiae). A process for producing vertebrate fusedpolypeptides is further provided and comprises culturing host cellsunder conditions suitable for expression of vertebrate fused andrecovering the same from the cell culture. The present inventionincludes the proteins and polypeptides with or without associatednative-pattern glycosylation. The recombinant proteins when expressed inyeast or mammalian expression systems (e.g., COS-7 cells) may be similaror significantly different in molecular weight and glycosylation patternfrom the corresponding native proteins. Expression of mammalian MCEMPsin bacterial expression systems, such as E. coli, providesnon-glycosylated molecules. Variant proteins comprising inactivatedN-glycosylation sites are also within the scope of the presentinvention. Such variants are expressed in a more homogeneous, reducedcarbohydrate form.

Recombinant Expression for MCEMPs

Isolated and purified recombinant MCEMPs are provided according to thepresent invention by incorporating the corresponding nucleotide sequenceinto expression vectors and expressing the nucleotide sequence insuitable host cells to produce the polypeptide.

Expression Vectors

Recombinant expression vectors containing a nucleotide sequence encodingthe polypeptide can be prepared using well known techniques. Theexpression vectors include a nucleotide sequence operably linked tosuitable transcriptional or translational regulatory nucleotidesequences such as those derived from mammalian, microbial, viral, orinsect genes. Examples of regulatory sequences include transcriptionalpromoters, operators, enhancers, mRNA ribosomnal binding sites, andappropriate sequences which control transcription and translationinitiation and termination. Nucleotide sequences are “operably linked”when the regulatory sequence functionally relates to the nucleotidesequence for the appropriate polypeptide. Thus, a promoter nucleotidesequence is operably linked to a MCEMP sequence if the promoternucleotide sequence controls the transcription of the appropriatenucleotide sequence.

The ability to replicate in the desired host cells, usually conferred byan origin of replication and a selection gene by which transformants areidentified, may additionally be incorporated into the expression vector.

In addition, sequences encoding appropriate signal peptides that are notnaturally associated with MCEMPs can be incorporated into expressionvectors. For example, a nucleotide sequence for a signal peptide(secretory leader) may be fused in-frame to the polypeptide sequence sothat the polypeptide is initially translated as a fusion proteincomprising the signal peptide. A signal peptide that is functional inthe intended host cells enhances extracellular secretion of theappropriate polypeptide. The signal peptide may be cleaved from thepolypeptide upon secretion of polypeptide from the cell.

Host Cells

Suitable host cells for expression of MCEMPs include prokaryotes, yeast,archae, and other eukaryotic cells. Appropriate cloning and expressionvectors for use with bacterial, fungal, yeast, and mammnalian cellularhosts are well known in the art, e.g., Pouwels et al. Cloning Vectors: ALaboratory Manual, Elsevier, N.Y. (1985). The vector may be a plasmidvector, a single or double-stranded phage vector, or a single ordouble-stranded RNA or DNA viral vector. Such vectors may be introducedinto cells as polynucleotides, preferably DNA, by well known techniquesfor introducing DNA and RNA into cells. The vectors, in the case ofphage and viral vectors also may be and preferably are introduced intocells as packaged or encapsulated virus by well known techniques forinfection and transduction. Viral vectors may be replication competentor replication defective. In the latter case viral propagation generallywill occur only in complementing host cells. Cell-free translationsystems could also be employed to produce the protein using RNAs derivedfrom the present DNA constructs.

Prokaryotes useful as host cells in the present invention include gramnegative or gram positive organisms such as E. coli or Bacilli. In aprokaryotic host cell, a polypeptide may include a N-terminal methionineresidue to facilitate expression of the recombinant polypeptide in theprokaryotic host cell. The N-terminal Met may be cleaved from theexpressed recombinant MCEMPs. Promoter sequences commonly used forrecombinant prokaryotic host cell expression vectors include β-lactamaseand the lactose promoter system.

Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids such as the cloningvector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin andtetracycline resistance and thus provides simple means for identifyingtransformed cells. To construct an expression vector using pBR322, anappropriate promoter and a DNA sequence are inserted into the pBR322vector. Other commercially available vectors include, for example,pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), pGEM1 (PromegaBiotec, Madison, Wis., USA), and the pET (Novagen, Madison, Wis., USA)and pRSET (Invitrogen Corporation, Carlsbad, Calif., USA) series ofvectors (Studier, F. W., J. Mol. Biol. 219: 37 (1991); Schoepfer, R.Gene 124: 83 (1993)).

Promoter sequences commonly used for recombinant prokaryotic host cellexpression vectors include T7, (Rosenberg, A. H., Lade, B. N., Chui,D-S., Lin, S-W., Dunn, J. J., and Studier, F. W. (1987) Gene (Amst.) 56,125-135), β-lactamasc (penicillinase), lactose promoter system (Chang etal., Nature 275:615, (1978); and Goeddel et al., Nature 281:544,(1979)), tryotophan (trp) promoter system (Goeddel et al., Nucl. AcidsRes. 8:4057, (1980)), and tac promoter (Maniatis, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, p.412 (1982)).

Yeasts useful as host cells in the present invention include those fromthe genus Saccharomyces, Pichia, K. Actinomycetes and Kluyveromyces.Yeast vectors will often contain an origin of replication sequence froma 2μ yeast plasmid, an autonomously replicating sequence (ARS), apromoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem. 255:2073, (1980)) or other glycolytic enzymes (Holland etal., Biochem. 17:4900, (1978)) such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other suitable vectors andpromoters for use in yeast expression are further described in Fleer etal., Gene, 107:285-195 (1991). Other suitable promoters and vectors foryeast and yeast transformation protocols are well known in the art.

Yeast transformation protocols are known to those of skill in the art.One such protocol is described by Hinnen et al., Proceedings of theNational Academy of Sciences USA, 75:1929 (1978). The Hinnen protocolselects for Trp.sup.+transformants in a selective medium, wherein theselective medium consists of 0.67% yeast nitrogen base, 0.5% casaminoacids, 2% glucose, 10 μg/ml adenine, and 20 μg/ml uracil.

Mammalian or insect host cell culture systems well known in the artcould also be employed to express recombinant MCEMPs, e.g., Baculovirussystems for production of heterologous proteins in insect cells (Luckowand Summers, Bio/Technology 6:47 (1988)) or Chinese hamster ovary (CHO)cells for mammalian expression may be used. Transcriptional andtranslational control sequences for mammalian host cell expressionvectors may be excised from viral genomes. Commonly used promotersequences and enhancer sequences are derived from Polyoma virus,Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus. DNAsequences derived from the SV40 viral genome may be used to provideother genetic elements for expression of a structural gene sequence in amammalian host cell, e.g., SV40 origin, early and late promoter,enhancer, splice, and polyadenylation sites. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment which may also contain a viral origin ofreplication. Exemplary expression vectors for use in mammalian hostcells are well known in the art.

MCEMPs may, when beneficial, be expressed as a fusion protein that hasthe MCEMP attached to a fusion segment. The fusion segment often aids inprotein purification, e.g., by permitting the fusion protein to beisolated and purified by affinity chromatography. Fusion proteins can beproduced by culturing a recombinant cell transformed with a fusionnucleic acid sequence that encodes a protein including the fusionsegment attached to either the carboxyl and/or amino terminal end of theprotein. Preferred fusion segments include, but are not limited to,glutathione-S-transferase, β-galactosidase, a poly-histidine segmentcapable of binding to a divalent metal ion, and maltose binding protein.

Since the MCEMPs lack a discernable leader peptide, a heterologoussignal peptide may be advantageously fused to the N-terminus of asoluble MCEMP to promote secretion thereof. The signal peptide can becleaved from the protein upon secretion from the host cell. The need tolyse the cells and recover the recombinant soluble protein from thecytoplasm thus is avoided. In one embodiment of the invention, a solublefusion protein comprises a first polypeptide derived from theextracellular domain of MCEMP1 fused to a second polypeptide added forpurposes such as facilitating purification or effecting dimer formation.Suitable second polypeptides do not inhibit secretion of the solublefusion protein. Examples of soluble polypeptides include thosecomprising the entire extracellular domain. Representative examples ofthe soluble proteins of the present invention include, but are notlimited to, a polypeptide comprising amino acids of SEQ ID NO:2, whereinthe polypeptide is selected from amino acids 1 through 82 of SEQ IDNO:2, amino acids 6 through 65 of SEQ ID NO:2, or any fragment thereofthat retains the ability to bind MCEMP1 ligand. Truncated forms of theinventive proteins, including soluble polypeptides, may be prepared byany of a number of conventional techniques.

Expression and Recovery

According to the present invention, isolated and purified MCEMPs may beproduced by the recombinant expression systems described above. Themethod comprises culturing a host cell transformed with an expressionvector comprising a nucleotide sequence that encodes the polypeptideunder conditions sufficient to promote expression of the polypeptide.The polypeptide is then recovered from culture medium or cell extracts,depending upon the expression system employed. As is known to theskilled artisan, procedures for purifying a recombinant polypeptide willvary according to such factors as the type of host cells employed andwhether or not the recombinant polypeptide is secreted into the culturemedium. When expression systems that secrete the recombinant polypeptideare employed, the culture medium first may be concentrated. Followingthe concentration step, the concentrate can be applied to a purificationmatrix such as a gel filtration medium. Alternatively, an anion exchangeresin can be employed, e.g., a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose, or other types commonly employed in proteinpurification. Also, a cation exchange step can be employed. Suitablecation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. Further, one or more reversed-phasehigh performance liquid chromatography (RP-HPLC) steps employinghydrophobic RP-HPLC media (e.g., silica gel having pendant methyl orother aliphatic groups), ion cxchange-HPLC (e.g., silica gel havingpendant DEAE or sulfopropyl (SP) groups), or hydrophobicinteraction-HPLC (e.g., silica gel having pendant phenyl, butyl, orother hydrophobic groups) can be employed to further purify the protein.Some or all of the foregoing purification steps, in variouscombinations, are well known in the art and can be employed to providean isolated and purified recombinant polypeptide.

Recombinant polypeptide produced in bacterial culture is usuallyisolated by initial disruption of the host cells, centrifugation,extraction from cell pellets if an insoluble polypeptide, or from thesupernatant fluid if a soluble polypeptide, followed by one or moreconcentration, salting-out, ion exchange, affinity purification, or sizeexclusion chromatography steps. Finally, RP-HPLC can be employed forfinal purification steps. Microbial cells can be disrupted by anyconvenient method, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Agonists and Antagonists Screening

In another aspect, the present invention provides a screening method foridentifying mast cell-expressed membrane protein agonists andantagonists. The screening method comprises exposing a mastcell-expressed membrane protein to a potential mast cell-expressedmembrane protein agonist/antagonist and determining whether thepotential agonist/antagonist interacts with the protein. If thepotential agonist/antagonist interacts with the protein, particularly bybinding to the protein, there is a strong presumption that the potentialagonist/antagonist will actually function as an agonist or antagonistwhen administered in vivo to a patient and exposed to the native mastcell-expressed membrane protein. The agonists and antagonists identifiedusing the method can be characterized as an agonist or an antagonist byexposing mast cells capable of producing mediators to theagonist/antagonist and measuring mast cell degranulation. Agonists willincrease degranulation; antagonists will decrease degranulation. Anothermethod for screening comprises transfecting the cells with a reportergene constructs that contains MCEMP DNA binding sequences. Preferably,the potential agonist/antagonist is an organic compound or polypeptide,including antibodies. The screening methods are useful for identifyingcompounds that may function as drugs for preventing or treatingdiseases, particularly diseases characterized by relatively low orrelatively high cytokine production compared to non-disease states.

Adverse Side Effect Screening

In a further aspect, the present invention provides a screening methodfor determining whether pharmaceuticals are likely to cause undesirableside effects associated with reducing or increasing mast cell activity,particularly degranulation, when administered to an animal for thedesired indication. The screening method comprises exposing mast cellsexpressing MCEMP or a purified MCEMP to the pharmaceutical anddetermining whether the pharmaceutical interacts with the protein ormimics the biological function of the protein ligand. If thepharmaceutical interacts with MCEMPs, there is a likelihood that thepharmaceutical will cause adverse side effects when administered to ananimal for the desired indication. The adverse side effects result froman undesirable change in mast cell function or activity, particularlyunwanted degranulation. Pharmaceuticals that can be screened by thismethod include, but are not limited to, polypeptides, proteins,peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides,oligosaccharides, nucleotides, organic molecules, bioorganic molecules,peptidomimetics, pharmacological agents and their metabolites, andtranscriptional and translation control sequences. In a preferredembodiment, antibodies to be administered for a particular indicationare screened to see if they cross-react with MCEMPs and are thereforelikely to cause unwanted side effects when administered for the intendedindication.

MCEMP Expression Modulation

In yet another aspect, the present invention provides a method forblocking or modulating the expression of MCEMPs by interfering with thetranscription or translation of a DNA or RNA polynucleotide encoding theproteins. The method comprises exposing a cell capable of expressingMCEMPs to a molecule that interferes with the proper transcription ortranslation of a DNA or RNA polynucleotide encoding the protein. Themolecule can be an organic molecule, a bioorganic molecule, an antisensenucleotide, a RNAi nucleotide, or a ribozyme.

In a preferred embodiment, the method comprises blocking or modulatingthe expression of MCEMPs by exposing a cell to a polynucleotide that isantisense to or forms a triple helix with MCEMP-encoding DNA or with DNAregulating expression of MCEMP-encoding DNA. The cell is exposed toantisense polynucleotide or triple helix-forming polynucleotide in anamount sufficient to inhibit or regulate expression of the proteins.Also, the present invention provides a method for blocking or modulatingexpression of MCEMPs in an animal by administering to the animal apolynucleotide that is antisense to or forms a triple helix withMCEMP-encoding DNA or with DNA regulating expression of MCEMP-encodingDNA. The animal is administered antisense polynucleotide or triplehelix-forming polynucleotide in an amount sufficient to inhibit orregulate expression of MCEMPs in the animal. Preferably, the antisensepolynucleotide or triple helix-forming polynucleotide is a DNA or RNApolynucleotide.

Methods for exposing cells to antisense polynucleotides and foradministering antisense polynucleotides to animals are well known in theart. In a preferred method, the polynucleotide is incorporated into thecellular genome using know methods and allowed to be expressed insidethe cell. The expressed antisense polynucleotide binds topolynucleotides coding for MCEMPs and interferes with theirtranscription or translation.

The methods are useful for inhibiting MCEMP expression while conductingresearch on various types of cells, e.g., mast cells or lung cells, andfor preventing or treating animal disease characterized by excesscellular activity, particularly degranulation, compared to non-diseasestates.

Disease Predisposition Diagnostic

In another aspect, the present invention provides a method fordiagnosing the predisposition of a patient to develop diseases caused byunwanted activity of cells expressing MCEMPs. The invention is basedupon the discovery that the presence of or increased amount of MCEMPs incertain patient cells, tissues, or body fluids indicates that thepatient is predisposed to certain immune diseases. In one embodiment,the method comprises collecting a cell, tissue, or body fluid sampleknown to contain few if any MCEMPs from a patient, analyzing the tissueor body fluid for the presence of MCEMPs in the tissue, and predictingthe predisposition of the patient to certain immune diseases based uponthe presence of MCEMPs in the tissue or body fluid. In anotherembodiment, the method comprises collecting a cell, tissue, or bodyfluid sample known to contain a defined level of MCEMPs from a patient,analyzing the tissue or body fluid for the amount of MCEMPs in thetissue, and predicting the predisposition of the patient to certainimmune diseases based upon the change in the amount of MCEMPs in thetissue or body fluid compared to a defined or tested level establishedfor normal cell, tissue, or bodily fluid. The defined level of MCEMPsmay be a known amount based upon literature values or may be determinedin advance by measuring the amount in normal cell, tissue, or bodyfluids. Specifically, determination of MCEMPs levels in certain tissuesor body fluids permits specific and early, preferably before diseaseoccurs, detection of immune diseases in the patient. Immune diseasesthat can be diagnosed using the present method include, but are notlimited to, the immune diseases described herein. In the preferredembodiment, the tissue or body fluid is mast cells and lung tissue.

Disease Prevention and Treatment

In another aspect, the present invention provides a method forpreventing or treating mast cell mediated diseases in a mammal. Themethod comprises administering a disease preventing or treating amountof a MCEMP agonist or antagonist to the mammal. The agonist orantagonist binds to MCEMP or its ligand and regulates the activity ofthe cell, particularly degranulation of mast cells, to produce mast cellmediator levels characteristic of non-disease states. Preferably, thedisease is an allergy, asthma, autoimmune, or other inflammatorydisease. Most preferably, the disease is an allergy or asthma.

The dosages of MCEMP agonist or antagonist vary according to the age,size, and character of the particular mammal and the disease. Skilledartisans can determine the dosages based upon these factors. The agonistor antagonist can be administered in treatment regimes consistent withthe disease, e.g., a single or a few doses over a few days to amelioratea disease state or periodic doses over an extended time to preventallergy or asthma.

The agonists and antagonists can be administered to the mammal in anyacceptable manner including by injection, using an implant, and thelike. Injections and implants are preferred because they permit precisecontrol of the timing and dosage levels used for administration. Theagonists and antagonists are preferably administered parenterally. Asused herein parenteral administration means by intravenous,intramuscular, or intraperitoneal injection, or by subcutaneous implant.

When administered by injection, the agonists and antagonists can beadministered to the mammal in a injectable formulation containing anybiocompatible and agonists and antagonists compatible carrier such asvarious vehicles, adjuvants, additives, and diluents. Aqueous vehiclessuch as water having no nonvolatile pyrogens, sterile water, andbacteriostatic water are also suitable to form injectable solutions. Inaddition to these forms of water, several other aqueous vehicles can beused. These include isotonic injection compositions that can besterilized such as sodium chloride, Ringer's, dextrose, dextrose andsodium chloride, and lactated Ringer's. Nonaqueous vehicles such ascottonseed oil, sesame oil, or peanut oil and esters such as isopropylmyristate may also be used as solvent systems for the compositions.Additionally, various additives which enhance the stability, sterility,and isotonicity of the composition including antimicrobialpreservatives, antioxidants, chelating agents, and buffers can be added.Any vehicle, diluent, or additive used would, however, have to bebiocompatible and compatible with the agonists and antagonists accordingto the present invention.

MCEMP Polypeptide Diagnostic

The antibodies of the present invention may also be used in a diagnosticmethod for detecting MCEMPs expressed in specific cells, tissues, orbody fluids or their components. The method comprises exposing cells,tissues, or body fluids or their components to an antibody of thepresent invention that binds to a MCEMP and determining if the cells,tissues, or body fluids or their components bind to the antibody. Cells,tissues, or body fluids or their components that bind to the antibodycells, tissues, or body fluids or their components that bind to theantibody are diagnosed as cells, tissues, or body fluids that containMCEMPs. Such method is useful for determining if a particular cell,tissue, or body fluid is one of a certain type of cell, tissue, or bodyfluid previously known to contain MCEMPs. Various diagnostic methodsknown in the art may be used, e.g., competitive binding assays, director indirect sandwich assays, and immunoprecipitation assays conducted ineither heterogeneous or homogeneous phases.

Rather than using monoclonal antibodies in diagnostic kits, it may bepossible to detect the presence of MCEMPs by using other compounds thatbind to it, wherein the compounds are labeled and able to be detected.Such compounds may be isolated by screening compound libraries and/orpeptide libraries. Library members, which are capable of interactingwith MCEMPs, can be labeled with a fluorescent marker, or a radioactivemarker, using a linker such as a peptide or other covalent chemicalconjugate to join the compound with the marker. The resulting labeledcompound can be used in a diagnostic kit to indicate the presence ofMCEMP positive cells, using well-known methods.

MCEMP Polypeptide Purification

The antibodies of the present invention may also be used in a method forisolating and purifying MCEMPs from recombinant cell cultures,contaminants, and native environments. The method comprises exposing acomposition containing MCEMPs and contaminants to an antibody capable ofbinding to the MCEMPs, allowing the MCEMPs to bind to the antibody,separating the antibody-MCEMP complexes from the contaminants, andrecovering the MCEMPs from the complexes. Various purification methodsknown in the art may be used, e.g., affinity purification methods thatrecover MCEMPs from recombinant cell culture or native sources. In thismethod, the antibodies that select MCEMPs are immobilized on a suitablesupport such a Sephadex resin or filter paper using methods well knownin the art. The immobilized antibody then is contacted with a samplecomposition containing the MCEMPs to be purified and contaminants. Thesupport is then washed with a suitable solvent capable of removingsubstantially all the material in the sample except the MCEMPs bound tothe immobilized antibody. Finally, the support is washed with anothersuitable solvent that that removes the MCEMPs from the antibody.

Knockout Animals

In another aspect, the present invention provides a knockout animalcomprising a genome having a heterozygous or homozygous disruption inits endogenous MCEMP gene that suppresses or prevents the expression ofbiologically functional MCEMPs. Preferably, the knockout animal of thepresent invention has a homozygous disruption in its endogenous MCEMPgene. Preferably, the knockout animal of the present invention is amouse. The knockout animal can be made easily using techniques known toskilled artisans. Gene disruption can be accomplished in several waysincluding introduction of a stop codon into any part of the polypeptidecoding sequence that results in a biologically inactive polypeptide,introduction of a mutation into a promoter or other regulatory sequencethat suppresses or prevents polypeptide expression, insertion of anexogenous sequence into the gene that inactivates the gene, and deletionof sequences from the gene.

Several techniques are available to introduce specific DNA sequencesinto the mammalian germ line and to achieve stable transmission of thesesequences (transgenes) to each subsequent generation. The most commonlyused technique is direct microinjection of DNA into the pronucleus offertilized oocytes. Mice or other animals derived from these oocyteswill be, at a frequency of about 10 to 20%, the transgenic founders thatthrough breeding will give rise to the different transgenic mouse lines.Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art, e.g., U.S. Pat. Nos. 4,736,866, 4,870,009, and4,873,191 and in Hogan, B., Manipulating the Mouse Embryo, (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similarmethods are used for production of other transgenic animals.

Embryonic stem cell (“ES cell”) technology can be used to createknockout mice (and other animals) with specifically deleted genes.Totipotent embryonic stem cells, which can be cultured in vitro andgenetically modified, are aggregated with or microinjected into mouseembryos to produce a chimeric mouse that can transmit this geneticmodification to its offspring. Through directed breeding, a mouse canthus be obtained that lacks this gene. Several other methods areavailable for the production of genetically modified animals, e.g., theintracytoplasmic sperm injection technique (ICSI) can be used fortransgenic mouse production. This method requires microinjecting thehead of a spermatocyte into the cytoplasm of an unfertilized oocyte,provoking fertilization of the oocyte, and subsequent activation of theappropriate cellular divisions of a preimplantation embryo. The mouseembryos thus obtained are transferred to a pseudopregnant receptorfemale. The female will give birth to a litter of mice. In ICSI appliedto transgenic mouse production, a sperm or spermatocyte heads suspensionis incubated with a solution containing the desired DNA molecules(transgene). These interact with the sperm that, once microinjected, actas a carrier vehicle for the foreign DNA. Once inside the oocyte, theDNA is integrated into the genome, giving rise to a transgenic mouse.This method renders higher yields (above 80%) of transgenic mice thanthose obtained to date using traditional pronuclear microinjectionprotocols.

Vaccines

In another aspect, the present invention provides a vaccine useful forimmunizing a mammal against mast cell or other MCEMP mediated diseasescomprising a pharmaceutically acceptable carrier and one or more MCEMPsor immunogenic fragments thereof. The vaccine is administrated tomammals suffering from or susceptible to MCEMP mediated diseases. Thevaccine induces the formation of antibodies in the immunized mammal thatinteract with MCEMPs and regulate the activity and function of cellsexpressing MCEMPs, including regulating the concentration of mast cellsor other MCEMP expressing cells. The vaccine can contain one or moreMCEMPs or immunogenic fragments alone or in combination with suitableadjuvants and/or other antigens and therapeutics.

In a further aspect, the present invention provides a method forimmunizing a mammal against mast cell or other MCEMP mediated diseasescomprising injecting one or more MCEMPs or immunogenic fragments thereofinto the mammal. The MCEMP or immunogenic fragment can be injected aloneor in combination with suitable adjuvants and/or other antigens andtherapeutics.

Generally, antigens are presented to the immune system using majorhistocompatibility complex (MHC) molecules, i.e., MHC Class I moleculesand MHC Class II molecules. Endogenous or self antigens, such as MCEMPs,are usually bound to MHC class I molecules and presented to cytotoxic Tcells (“CTL(s)”). Exogenous antigens, such as viral antigens, areusually bound to MHC Class II molecules and presented to T cells thatinteract with B cells to produce antibodies.

Antigens presented via the Class II pathway, known as MHC ClassII-restricted antigens or Class II antigens, are recognized by andactivate T cells. These activated T cells cause a complete immuneresponse to the Class II antigens. Because self antigens normally arenot presented to the immune system through the MHC Class II pathway, theimmune system does not recognize these self antigens as foreign and doesnot form a complete immune response to such antigens.

In one embodiment, a MCEMP antigen is injected in combination,simultaneously or contemporaneously, with other antigens that aredesigned to stimulate or manipulate the immune response. Preferably, theMCEMP antigen is injected as part of a construct comprising the MCEMPantigen and other antigens that are designed to induce a cellular immuneresponse. Such other antigens are designed to enhance antigenpresentation to T cells and induce a more potent immune response toantigens such as MCEMP that typically elicit an incomplete immuneresponse because they are not recognized by the immune system as foreignantigens.

Typically, MCEMP is injected in combination with Class II antigens. Useof other antigens to stimulate the immune system via the MHC Class IIpathway in combination with the MCEMP antigen, which may be recognizedby the immune system as a self antigen that elicits a weak or incompleteimmune response, helps to ensure that the MCEMP antigen will be treatedby the immune system as a foreign antigen that elicits a complete immunesystem response. Preferably, the MCEMP antigen and the Class II antigenare part of a construct wherein the antigens are part of a singlemolecule. In another aspect, the present invention provides a constructcomprising a MCEMP antigen and another antigen in a single molecule.Preferably, the other antigen is a Class II antigen.

In another aspect, the present invention provides a vaccine useful forimmunizing a mammal against mast cell or other MCEMP mediated diseasescomprising a pharmaceutically acceptable carrier and a vector containinga nucleic acid sequence encoding a MCEMP or antigenic fragment thereof.Preferably, the vaccine comprises a nucleotide sequence selected fromthe group consisting of SEQ ID NO:1; a variant of SEQ ID NO:1; and afragment of SEQ ID NO:1. Most preferably, the vaccine comprises thenucleotide sequence that encodes the MCEMP having the sequence shown inSEQ ID NO:2 (“MCEMP1”) or antigenic fragment thereof, particularly anantigenic fragment comprising the extracellular domain (amino acids 106through 187) or an antigenic fragment thereof.

The nucleotide vaccines of the present invention are useful forpreventing or treating a disease caused by a malfunction of the immunesystem in distinguishing self from non-self. The vaccines cause theimmune system to elicit self protective immunity and thus limit its ownharmful activity to times when such a response is needed. In particular,DNA vaccines represent a novel means of expressing antigens in vivo forthe generation of both humoral and cellular immune responses. Thistechnology has proven successful in obtaining immunity not only toforeign antigens and tumors, but also to self antigens, such as a T cellreceptor genes or autologous cytokines. Since DNA vaccines elicit bothcellular and humoral responses against products of a given construct,the vaccines can be a very effective tool in eradicating diseased orunwanted cells. The direct injection of gene expression cassettes into aliving host transforms a number of cells into factories for productionof the introduced gene products. Expression of these delivered genes hasimportant immunological consequences and may result in the specificimmune activation of the host against the novel expressed antigens. Thisunique approach to immunization can overcome deficits of traditionalantigen-based approaches and provide safe and effective prophylactic andtherapeutic vaccines. The host normal cells (nonhemopoietic) can expressand present MCEMP antigens to the immune system. The transfected cellsdisplay fragments of the antigens on their cell surfaces together withclass I or class II major hisotcompatibility complexes (MHC I or MHCII). The MHC I display acts as a distress call for cell-mediated immuneresponse, which dispatches CTLs that destroy the transfected cells. Ingeneral, when a cytopathic virus infects a host normal cell, the viralproteins are endogenously processed and presented on the cell surface,or in fragments by MHC molecules. Foreign defined nucleic acidtransfected and expressed by normal cells can mimic viral infections.

An immunogenic fusion polypeptide encoded on a vector as describedherein comprises a T cell epitope portion and a B cell epitope portion.A T cell epitope portion encoded on the vector comprises a broad rangeor “universal” helper T cell epitopes that bind the antigen presentingsite of multiple (i.e., 2, 3, 4, 5, 6 or more) class II majorhistocompatibility (MHC) molecules and can form a tertiary complex witha T cell antigen receptor, i.e., MHC:antigen:T cell antigen receptor. A“non-endogenous protein” is a protein that is not endogenous to themammal to be treated. Such non-endogenous proteins, or fragmentsthereof, useful as T cell epitope portions of the immunogenic fusionpolypeptide include tetanus toxoid; diphtheria toxin; class IIMHC-associated invariant chain; influenza hemagglutinin T cell epitope;keyhole limpet hemocyanin (KLH); a protein from known vaccines includingpertussis vaccine, the Bacile Calmette-Guerin (BCG) tuberculosisvaccine, polio vaccine, measles vaccine, mumps vaccine, rubella vaccine,and purified protein derivative (PPD) of tuberculin; and also syntheticpeptides which bind the antigen presenting site of multiple class IIhistocompatibility molecules, such as those containing natural aminoacids described by Alexander et al. (Immunity, 1: 751-761 (1994)). Whenattached to a MCEMP epitope portion, the T cell epitope portion enablesthe immunogenic fusion polypeptide to break tolerance and permitantibodies to be made that react with endogenous MCEMPs. By “breakingtolerance” is meant forcing an organism to mount an immune response to aprotein, such as endogenous MCEMPs, that the organism does not normallyfind immunogenic.

DNA vaccines recently have been shown to be a promising approach forimmunization against a variety of infectious diseases. Michel, M L etal., Huygen, K, et al., and Wang, B, et al. Delivery of naked DNAscontaining microbial antigen genes can induce antigen-specific immuneresponses in the host. The induction of antigen-specific immuneresponses using DNA-based vaccines has shown some promising effects.Wolff, J. A., et al. Recent studies have demonstrated the potentialfeasibility of immunization using a DNA-mediated vaccine for CEA andMUC-1. Conry, R. M., et al. and Graham, R. A., et al.

DNA-based vaccination has been shown to have a greater degree of controlof antigen expression, toxicity, and pathogenicity than live attenuatedvirus immunization. The construction, operation, and use of the abovepharmaceutically acceptable carriers for DNA vaccination and the abovedelivery vehicles are described in detail in U.S. Pat. No. 5,705,151 toDow et al., entitled “Gene Therapy for T Cell Regulation”, which isdirected at anti-cancer treatment, and is hereby incorporated byreference as if fully set forth herein.

In a further aspect, the present invention provides a method forimmunizing a mammal against mast cell or other MCEMP mediated diseasescomprising injecting a pharmaceutically acceptable carrier and a vectorcontaining a nucleic acid sequence encoding a MCEMP or antigenicfragment thereof. Preferably, the method comprises injecting a vaccinecomprising a nucleotide sequence selected from the group consisting ofSEQ ID NO:1; a variant of SEQ ID NO:1; and a fragment of SEQ ID NO:1.Most preferably, the vaccine comprises the nucleotide sequence thatencodes the MCEMP having the sequence shown in SEQ ID NO:2 (“MCEMP1”) orantigenic fragment thereof, particularly an antigenic fragmentcomprising the extracellular domain (amino acids 106 through 187) or anantigenic fragment thereof.

Identification of MCEMP1

MCEMP1 was identified by subtractive hybridization using human mast cellmRNA as a tester and a combination of mRNAs from human THP-1 (˜45%),Daudi (˜35%) and TF-1 (˜20%) cell lines as drivers. Approximately 45subtracted clones were isolated, sequenced, and used to search formatches in the publicly available nucleotide/protein databases. A cDNAclone comprising a 369 base pair (bp) insert isolated by the subtractivehybridization only matched to a number of EST clones that containpartial cDNA sequences, but it showed no significant homology to anycDNA sequences that encode known or predicted proteins in the GenBankdatabase.

Two oligonucleotide primers: 5′ CTCCCAGAAAGGTGATGAA 3′ (SEQ ID NO: 3)and 5′ TAGACAGAAAACACGCCGCAGTA 3′ (SEQ ID NO: 4)based on the 369 bp insert sequence were synthesized and used to screena human peripheral blood leukocyte cDNA library (OriGene Technologies,Inc., Rockville, Md.). Several cDNA clones were isolated and sequenced.Three alternative splicing forms of mRNAs were identified by comparingthe cDNA sequences with genomnic sequences in the GenBank database. Twoof cDNA clones represent aberrant mRNA transcripts, because the putativetranslation product in all three reading frames would be aborted by stopcodons. However, the majority of the cDNA clones predicted a proteinproduct, which was derived from seven exons in the MCEMP1 gene. One suchcDNA clone (9E) contained a full length coding region (564 bp) about 450bp 5′ untranslated region and about 726 bp 3′ untranslated region.

In addition, a cDNA clone was obtained from a HMC-1 cell line by RT-PCRusing an oligo primer covering the starting methionine codon 5′GACCATGGAAGTGGAGGAAATCTAC 3′ (SEQ ID NO:5) and an oligo primer coveringthe stop codon, 5′ GCAGGTGCAGCCCCATCTT 3′ (SEQ ID NO:6). These cDNAsencoded a polypeptide of 187 amino acids. The predicted startingmethionine codon was associated with a perfect Kozak sequence motif(ACCATGG), making it optimal for translation initiation. An allelicvariation was found at amino acid residue 167 (Ile < >Val) among thecDNA clones, which was caused by a single nucleotide change at the firstcodon position (ATT < >GTT).

Computer-assisted analysis predicted that MCEMP1 had a transmembranesequence located at amino acid residues 83 to 105. There did not appearto be a discernable N-terninal hydrophobic leader sequence. Thepredicted molecular mass for MCEMP1 was 21 kDa. A comparison of bothnucleotide and amino acid sequences with GenBank or European MolecularBiology Laboratory databases revealed that it shared a 37% amino acididentity with BAB25183, a putative mouse sequence identified by TheRIKEN Genome Exploration Research Group Phase II Team and the FANTOMConsortium. A 3-dimensional structure prediction was carried out using athreading-based fold recognition method (Kelley et al., J. Mol. Biol.299:499-520 (2000)). Briefly, using a library of known proteinstructures, the MCEMP1 sequence was “threaded” and scored forcompatibility. Four components were used in the scoring system: 1D and3D sequence profiles coupled with secondary structure and salvationpotential information. Since the prediction of transmembrane helixshowed that MCEMP1 contained a transmembrane segment (amino acids 83through 105), fold recognition process has been applied on theN-terminal part (amino acids 1 through 82) and C-terminal part (aminoacids 106 through 187) separately to improve the accuracy. The resultsshowed that the N-terminal region (amino acids 6 through 65) likelyadopts an Ig-like β sandwich fold, sharing 21% identity with Ig domainof mouse T-cell receptor α-chain.

EXAMPLES

This invention can be further illustrated by the following examples ofpreferred embodiments thereof, although it will be understood that theseexamples are included merely for purposes of illustration and are notintended to limit the scope of the invention unless otherwisespecifically indicated.

Experimental Methods Cell Culture

Human cord blood CD34⁺ cells (Bio-Whittaker, Walkersville, Md.) werecultured up to 9 weeks in culture media consisting of RPM11640(Invitrogen) supplemented with 20% FBS (Sigma-Aldrich, St. Louis, Mo.),2 mM L-glutamine, 50 μM 2-ME, 100 U/ml penicillin, 100 μg/mlstreptomycin, 10 μg/ml gentamicin, 80 ng/ml SCF, 50 ng/ml IL-6 and 5ng/ml IL-10. Cells were stained with anti-tryptase mAb to determine thepercentage of mast cells. Cell suspensions were seeded at a density of5×10⁵ cells/ml and cytokine-supplemented medium was replaced once aweek. Recombinant human IgE was used for IgE cross-linking experiment.Other cell lines were cultured following ATCC's recommendations.

Expression Construct and Transfection

Flag-tagged MCEMP1 cDNA was PCR-amplified by using two oligo primers: 5′CACCATGGACTACAAAGACGATGACGACAAGGAAGTGGAGGAAATCTACAAGC 3′ (SEQ ID NO: 7)and 5′ TTGAGGTGAGGACTGTGGCATTT 3′. (SEQ ID NO: 8)The PCR product was cloned into pcDNA3.1D/V5-His vector (Invitrogen).This yield the plasmid, MCEMP1-FV, that expresses MCEMP1 fusion proteinwith Flag tag at N terminus and V5 tag at C terminus. For the N-terminalregion of MCEMP1 and Fc γ1 fusion construct (MCEMP1T-Fcγ1), the regionencoding amino acid 1-83 was PCR-amplified, and joined to Fc γ1 codingregion by additional round of PCR (SOEing, Ho, S. et al. 1989, Gene 77:51-59). The coding region of the fusion protein was cloned intopSecTag/FRT/V5-His-TOPO (Invitrogen).Transient transfection was performed using Lipofectamine Plus system(Invitrogen). Twenty micrograms of plasmid DNA was transfected into 293Tcells in a 100 mm tissue culture dish; and 40 hours later, the cellswere harvested in PBS-based, enzyme-free cell dissociation buffer(Invitrogen) for protein analysis.

Protein Extraction and Western Blot Analysis

The whole cell protein sample was prepared by resuspending 3×10⁵ cellsin 100 μl of ddH₂O, and heated at 98° C. for 5 minutes after addingequal volume of 2× sample loading buffer. To separate membrane fractionfrom soluble fraction, 5×10⁵ cells were subject to lysis procedurethrough either homogenization or freeze-thaw cycles. For homogenization,cells were first incubated in 150 μl of ddH₂O for 10 minutes, thenpassed through a #22 syringe needle multiple times. Thereafter one tenthof 10× lysis buffer (200 mM Tris-HCl, pH7.6; 700 mM KCl; 50 mM EDTA) wasadded back and incubated for 5 minutes. For the freeze-thaw method,cells were suspended in 1× lysis buffer, and freeze-thawed three times.Insoluble membrane fraction was separated from soluble proteins bycentrifugation at maximum speed in a microcentrifuge.

The proteins were separated in a 15% SDS-PAGE. Western blot wasperformed as previously described [26] by using anti-Flag (Sigma) oranti-V5 mAb (Invitrogen).

Immunofluorescence Staining

The transfected 293T cells (1×10⁶) were washed and preincubated at 4° C.for 20 minutes in 100 μl of the enzyme-free cell dissociation buffer(Invitrogen) containing 1% BSA. Cells were then incubated withFITC-conjugated anti-Flag (20 μg/ml) (Sigma-Aldrich) or Anti-V5 mAb (10μg/ml) (Invitrogen) in the same buffer for 30 minutes. After threewashes, cells were resuspended in 100 μl of 1×PBS with 1%paraformaldehyde. Alternatively, human cord blood-derived mast cells,HMC-1 and THP-1 cells were incubated with anti-MCEMP1 monoclonalantibodies, then incubated with 2^(nd) antibody, FITC-conjugated goatanti-mouse IgG antibody. All samples were analyzed using FACScan (BectonDickinson, Franklin Lake, N.J.) and/or microscopy.

Generation of Anti-MCEMP1 mAbs

Mice were immunized by antigen display constructs that contain the N(amino acid 1-83) and C terminal coding regions (amino acid 105-187).Hybridoma clones were generated and screened as conventional methods. InELISA screening, we coated 96-well plate with either MCEMP1-FV or MCEMP1fusion protein, then incubated with the supernatant of hybridoma clones.Goat anti-mouse IgG was used as 2^(nd) antibody to develop the signal.

Immunoprecipitation of Biotinylated Membrane Protein

Cell surface membrane proteins were biotinylated in 10 mg/ml ofD-biotinoyl-e-aminocaproic acid-N-hydroxysuccinimide ester(Boehringer)/10 mM sodium borate, pH8.8/150 mM sodium chloride. Thecells were washed extensively, and lysed in 1×PBS with 0.5% NP40 andprotease inhibitor mix (Boehringer). Immunoprecipitation was performedusing anti-MCEMP1 mAb and Protein A-Sepharose beads (Amersham) followingmanufacturer's recommendations. The immunoprecipitated proteins wereseparated in SDS-PAGE and the biotinylated proteins were detected bystreptavidin-HRP and ECL Detection Reagents (Amersham).

Example 1 Quantitative Real-time PCR Analysis of MCEMP1 mRNA Expression

Two sets of oligonucleotide primers: 5′ AAGGTGATGAATGAATAGGACTGA 3′ (SEQID NO: 9) and 5′ CCACCGTGACATGCCGAGACT 3′ (SEQ ID NO: 10)were selected from the MCEMP1 nucleotide sequences using Primer Express2.0 (Applied Biosystems, Inc.) and were synthesized and used in RT-PCRreactions to monitor the expression of MCEMP1.

Real-time quantitative PCR was performed with the ABI Prism 7900(Applied Biosystems, Inc.) sequence detection system, using CYBR Greenreagents, according to the manufacture's instructions. RNAs wereisolated to measure the level of expression of MCEMP1 in the followingcells: Daudi (a B lymphoblast cell line derived from Burkitt's lymphoma,ATCC No. CCL-213), THP-1 (a monocytic leukemia cell line, ATCC No.TIB202), TF-1 (a myeloid progenitor cell line, ATCC No. CRL-2003),HMC-1, (a mast cell line); primary monocytes; primary B cells; primarybasophils; CD34+ progenitor cells; in vitro cultured cord blood derivedmast cells (CBMC) at week 5 and week 9; macrophages and macrophagesactivated by LPS; HPB-ALL, (a T cell leukemia cell line); primarylymphocytes; neutrophils; and primary human vascular endothelial cells(HUVAC).

Equal amounts of each of the RNAs from the cell lines indicated abovewere used as PCR templates in reactions to obtain the threshold cycle(C_(t)). The C_(t) was normalized using the known C_(t) from 18S RNAs toobtain ΔC_(t). To compare relative levels of gene expression of MCEMP1in different cell lines, ΔΔC_(t) values were calculated by using thelowest expression level as the base, which were then converted to realfold expression difference values. MCEMP1 mRNA was found to be expressedin week 5 and week 9 in vitro cultured mast cells. Moderate levels werefound in monocytes. Among the five human tissues examined, MCEMP1 washighly expressed in mast cells and lung cells but very little expressionwas observed in heart, liver, brain, trachea, and kidney.

Example 2 Expression of MCEMP1 Protein

To determine the MCEMP1 gene product, MCEMP1 cDNA was PCR-amplified byusing two oligo primers: 5′CACCATGGACTACAAAGACGATGACGACAAGGAAGTGGAGGAAATCTACAAGC 3′ (SEQ ID NO: 11)and 5′ TTGAGGTGAGGACTGTGGCATTT 3′ (SEQ ID NO: 12)were cloned into pcDNA3.1D/V5-His vector (Invitrogen) with a Flag tagsequence attached to the N-terminus of MCEMP1 and a V5 tag fused to theC-terminus. The resultant clone, pMCEMP1-FV, was transiently transfectedinto 293T cells. Forty hours after transfection, transfected cells wereharvested and separated into membrane and cytosolic fractions by eithera homogenization or freeze-thaw method. Western blot analysis wasperformed using anti-Flag or anti-V5 mAb and anti-mouse IgG conjugates.MCEMP1 was expressed as a predominant 35 kDa protein. Minor forms of 29and 32 kDa proteins were also present in MCEMP1 transfected cells. Thefact that all the protein bands were larger than the calculatedmolecular weight, 27 kDa (21 kDa plus 6 kDa of tag), implies that MCEMP1might be post-translationally modified, e.g., by glycosylation in 293Tcells. Fractionation of cells resulted in the presence of MCEMP1 in themembrane fraction, but very little was present in the cytosol.

Example 3 Administering MCEMP1-binding Molecules

The antagonistic or agonistic MCEMP1 binding molecules, such asantibodies and biologically active fragments thereof, of the presentinvention can be administered to patients in an appropriatepharmacological formulation by a variety of routes, including, but notlimited to, intravenous infusion, intravenous bolus injection, andintraperitoneal, intradermal, intramuscular, subcutaneous, intranasal,intratracheal, intraspinal, intracranial, and oral routes. Suchadministration enables them to bind to endogenous MCEMP1 andinhibit/stimulate the action MCEMP1. These antagonists can also blockthe binding of the natural ligand for MCEMP1.

The estimated dosage of such antibodies is between 10 and 500 μg/ml ofserum. The actual dosage can be determined in clinical trials followingthe conventional methodology for determining optimal dosages, i.e.,extrapolating a dosage range from in vitro and in vivo experiments, andthen administering various dosages within the range to determine whichis most effective.

Example 4 Subcellular Localization

To determine whether MCEMP1 is expressed as a type II transmembraneprotein, MCEMP1 FV-transfected cells were lysed by either homogenizationor freeze-thaw method and the membrane fraction (pellet) was separatedfrom the soluble fraction by centrifugation. MCEMP1 was present mainlyin the membrane fraction as detected by both anti-Flag and anti-V5 mAbs;very little was detected in soluble fraction. To further determinewhether the Flag- and V5-tagged MCEMP1 is expressed on the cell surfaceand its orientation in the membrane, MCEMP1 FV-transfected cells wereincubated in living condition with FITC-conjugated anti-Flag or anti-V5mAb. Fluorescence microscopy and flow cytometric analysis showed thatwhile anti-V5 mAb was bound to the MCEMP1-FV on the membrane, anti-FlagmAb did not bind to the membrane (Table 1). These results show thatMCEMP1 is a type II transmembrane protein with the C-terminus exposed tothe outside of the cellular membrane and the N-terminus to thecytoplasmic compartment.

Example 5 Characterization of Mouse Monoclonal Antibodies against MCEMP1

Mouse monoclonal antibodies (mAb) were generated against MCEMP1 andscreened by ELISA, FACS, and Western blot analysis. Three of the mAbwere characterized extensively. Antibody clone AZ1C11 bound to bothMCEMP1-FV and MCEMP1T-Fcγ1, while antibody clones AZ1A8 and AZ3H6 onlypositively bound to full length of the MCEMP1 fusion protein. This showsthat AZ1A8 and AZ3H6 specifically interact with C-terminal region ofMCEMP1 and clone AZ1C11 interact with the N-terminal region of MCEMP1.Immuno-fluorescent staining of THP-1 and 293 T cells transfected withMCEMP1-Fc fusion protein confirmed the above results, i.e. antibodyclones AZ1A8 and AZ3H6 bind to the C-terminal end of MCEMP1 in theliving cells and clone AZ1C11 did not bind to the living cell. However,in Western blot analysis, AZ1A8 and AZ3H6 did not bind to MCEMP1 butclone AZ1C11 did bind to MCEMP1.

Example 6 Expression and Detection of Native MCEMP1 in CordBlood-Derived Mast Cells (CBMC) and HMC-1 Cells

The real time RT-PCR analysis showed that MCEMP1 is differentiallyexpressed in mast cells as well as in two long term-cultured cell lines,HMC-1 and THP-1. The immuno-fluorescent staining of CBMC, HMC-1, andTHP-1 cells confirmed those results. The native MCEMP1 was detected byantibody clones AZ1A8 and AZ3H6 in those three types of cells but notdetected in other cells tested. The binding of the antibodies to CBMCand HMC-1 cells behaved in a dose-dependent manner, i.e., the moreantibody input resulted in bigger shift of the fluorescent intensity bythe antibody-stained cells. The expression of MCEMP1 in CBMC was furtherassessed by immunoprecipitation of biotinylated membrane protein. Aprotein with molecular weight of ˜21 kD was detected by an anti-MCEMP1antibody but not by any other antibodies tested. Because the detectedMCEMP1 has the same molecular weight as predicted based on amino acidsequence, the native MCEMP1 is not glycosylated. TABLE 1Immuno-fluorescent staining of 293T cells transfected with MCEMP1-FVAntibody Anti-V5 Anti-Flag Cell Status Alive Fixed Alive FixedpV252-FV + + − + pcDNA3.1 − − − −

In the specification, there have been disclosed typical prefefredembodiments of the invention and, although specific terms are employed,they are used in a generic and descriptive sense only and not forpurposes of limitation, the scope of the invention being set forth inthe following claims. Obviously many modifications and variations of thepresent invention are possible in light of the above teachings. It istherefore to be understood that within the scope of the appended claimsthe invention may be practiced otherwise than as specifically described.

1. A purified polypeptide comprising an amino sequence selected from thegroup consisting of: SEQ ID NO:2; a variant of SEQ ID NO:2; a fragmentof SEQ ID NO:2; an amino acid sequence encoded by an isolatedpolynucleotide comprising a nucleotide sequence selected from the groupconsisting of: SEQ ID NO:1; a variant of SEQ ID NO:1; and a fragment ofSEQ ID NO:1.
 2. The purified polypeptide of claim 1 wherein thepolypeptide is encoded by a fragment of SEQ ID NO:1 comprisingnucleotides 455 through
 1018. 3. The purified polypeptide of claim 1wherein the polypeptide is an agonist or antagonist that specificallybinds to a mast cell-expressed membrane protein or its ligand andinhibits or activates the protein's cellular function.
 4. The purifiedpolypeptide of claim 3 wherein the polypeptide is an antagonist selectedfrom the group consisting of soluble forms of mast cell-expressedmembrane proteins and soluble polypeptides derived from theextracellular domains of mast cell-expressed membrane proteins that arecapable of interfering with the ability of a mast cell-expressedmembrane protein to interact with its natural ligand.
 5. The purifiedpolypeptide of claim 4 comprising an amino acid sequence selected fromthe group consisting of amino acids 106 to 187 of SEQ ID NO:2 orantagonist fragments thereof.
 6. The purified polypeptide of claim 3wherein the agonist or antagonist is an antibody.
 7. The purifiedpolypeptide of claim 6 wherein the antibody is selected from the groupconsisting of polyclonal, monoclonal, humanized, human, bispecific, andheteroconjugate antibodies.
 8. The purified polypeptide of claim 6wherein the antibody is a monoclonal antibody.
 9. An isolatedpolynucleotde comprising a nucleotide sequence selected from the groupconsisting of: SEQ ID NO:1; a variant of SEQ I) NO:1; a fragment of SEQID NO: 1; a nucleotide sequence that encodes a polypeptide having theamino acid sequence selected from the group consisting of: SEQ ID NO:2;a variant of SEQ ID NO:2; a fragment of SEQ ID NO:2.
 10. The isolatedpolynucleotide of claim 9 comprising a nucleotide sequence that encodesa polypeptide having an amino acid sequence selected from the groupconsisting of amino acids 106 to 187 of SEQ ID NO:2 or fragmentsthereof.
 11. An expression vector comprising the nucleotide sequence ofclaim
 9. 12. An isolated host cell selected from the group consisting ofa host cell comprising the expression vector of claim 11; a host cellcomprising the nucleotide sequence of claim 9; and a host cellcomprising the nucleotide sequence of claim
 10. 13. A screening methodfor identifying mast cell-expressed membrane protein agonists andantagonists, comprising: exposing a mast cell-expressed membrane proteinto a potential mast cell-expressed membrane protein agonist antagonist;and determining whether the potential agonist/antagonist interacts withthe protein.
 14. A screening method for determining whetherpharmaceuticals are likely to cause undesirable side effects associatedwith reducing or increasing mast cell activity when administered to amammal for the desired indication, comprising: exposing mast cellsexpressing mast cell-expressed membrane proteins or a purified mastcell-expressed membrane protein to a pharmaceutical; and determiningwhether the pharmaceutical interacts with the protein or mimics thebiological function of the protein ligand.
 15. A method for blocking ormodulating the expression of a cellular mast cell-expressed membraneprotein by interfering with the transcription or translation of a DNA orRNA polynucleotide encoding the mast cell-expressed membrane proteincomprising exposing a cell capable of expressing a mast cell-expressedmembrane protein to a molecule that interferes with the transcription ortranslation of a DNA or RNA polynucleotide encoding the mastcell-expressed membrane protein.
 16. The method of claim 14 wherein themolecule is selected from the group consisting of antisense nucleotides,RNAi nucleotides, and ribozymes that interfere with the propertranscription or translation of a DNA or RNA polynucleotide encoding themast cell-expressed membrane protein.
 17. The method of claim 14 whereinthe molecule is an antisense nucleotide that interferes with the propertranscription or translation of a DNA or RNA polynucleotide encoding themast cell-expressed membrane protein.
 18. A method for diagnosing thepredisposition of a patient to develop diseases caused by unwantedactivity of cells expressing mast cell-expressed membrane proteins,comprising: collecting a cell, tissue, or body fluid sample known tocontain few if any mast cell-expressed membrane proteins from a patient;analyzing the tissue or body fluid for the presence of mastcell-expressed membrane proteins in the tissue; and predicting thepredisposition of the patient to certain immune diseases based upon thepresence of mast cell-expressed membrane proteins in the tissue or bodyfluid.
 19. The method of claim 18 wherein the cells expressing mastcell-expressed membrane proteins are mast cells and the unwantedactivity is mast cell degranulation.
 20. A method for diagnosing thepredisposition of a patient to develop diseases caused by unwantedactivity of cells expressing mast cell-expressed membrane proteins,comprising: collecting a cell, tissue, or body fluid sample known tocontain a defined level of mast cell-expressed membrane proteins from apatient; analyzing the tissue or body fluid for the amount of mastcell-expressed membrane protein in the tissue; and predicting thepredisposition of the patient to certain immune diseases based upon thechange in the amount of mast cell-expressed membrane protein in thetissue or body fluid compared to a defined or tested level establishedfor normal cell, tissue, or bodily fluids.
 21. The method of claim 20wherein the cells expressing mast cell-expressed membrane proteins aremast cells and the unwanted activity is mast cell degranulation.
 22. Amethod for preventing or treating mast cell-expressed membrane proteinmediated diseases in a mammal comprising administering a diseasepreventing or treating amount of a mast cell-expressed membrane proteinagonist or antagonist to the mammal.
 23. The method of claim 22 whereinthe mast cell-expressed membrane protein agonist or antagonist is anantibody.
 24. A diagnostic method for detecting mast cell-expressedmembrane proteins expressed in specific cells, tissues, or body fluids,comprising: exposing cells, tissues, or body fluids or their componentsto an antibody that binds to a mast cell-expressed membrane protein; anddetermining if the cells, tissues, or body fluids or their componentsbind to the antibody.
 25. A method for producing an antibody that bindsto mast cell-expressed membrane proteins, comprising a method selectedfrom the group consisting of using isolated mast cell-expressed membraneproteins or antigenic fragments thereof as an antigen; using host cellsthat express recombinant mast cell-expressed membrane proteins as anantigen; and using DNA expression vectors containing the mastcell-expressed membrane protein gene to express the mast cell-expressedmembrane protein as an antigen for producing antibodies.
 26. Theantibody produced using the method of claim
 25. 27. The antibody ofclaim 25 selected from the group consisting of polyclonal, monoclonal,humanized, human, bispecific, and heteroconjugate antibodies.
 28. Amethod for isolating and purifying mast cell-expressed membrane proteinsfrom recombinant cell culture, contaminants, and native environments,comprising: exposing a composition containing mast cell-expressedmembrane proteins and contaminants to an antibody capable of binding tothe mast cell-expressed membrane proteins; allowing the mastcell-expressed membrane proteins to bind to the antibody; separating theantibody-mast cell-expressed membrane protein complexes from thecontaminants; and recovering the mast cell-expressed membrane proteinsfrom the complexes.
 29. The method of claim 28 wherein the antibody isan antibody of claim
 25. 30. A transgenic knockout animal whose genomecomprises a heterozygous or homozygous disruption in its endogenous mastcell-expressed membrane protein gene that suppresses or prevents theexpression of biologically functional mast cell-expressed membraneproteins.
 31. A vaccine useful for immunizing a mammal against mast cellor other mast cell-expressed membrane protein mediated diseasescomprising a pharmaceutically acceptable carrier and one or more mastcell-expressed membrane proteins or immunogenic fragments thereof.
 32. Amethod for immunizing a mammal against mast cell or other mastcell-expressed membrane protein mediated diseases comprising injectingone or more mast cell-expressed membrane proteins or immunogenicfragments thereof into the mammal.
 33. A vaccine useful for immunizing amammal against mast cell or other mast cell-expressed membrane proteinmediated diseases comprising a pharmaceutically acceptable carrier and avector containing a nucleic acid sequence encoding a mast cell-expressedmembrane protein or antigenic fragment thereof.
 34. The vaccine of claim33 wherein the nucleic acid sequence is a sequence selected from thegroup consisting of SEQ ID NO:1; a variant of SEQ ID NO:1; and afragment of SEQ ID NO:1.
 35. A method for immunizing a mammal againstmast cell or other mast cell-expressed membrane protein mediateddiseases comprising injecting a pharmaceutically acceptable carrier anda vector containing a nucleic acid sequence encoding a mastcell-expressed membrane protein or antigenic fragment thereof.
 36. Themethod of claim 35 wherein the nucleic acid sequence is a sequenceselected from the group consisting of SEQ ID NO:1; a variant of SEQ IDNO:1; and a fragment of SEQ ID NO:1.